Only few published data are available on ticks and tick-borne zoonotic pathogens in Bolivia. To evaluate rickettsial seroprevalence and infection in dogs and ticks, during February-April 2007, we collected whole blood, sera, and ticks from dogs living in the rural, peri-urban, and urban areas of Cochabamba, Bolivia. Dog sera were subjected to enzyme-linked immunosorbent assay test to detect IgG antibodies against Rickettsia rickettsii and 68.2% of samples were found to be positive (n = 30; 95% confidence interval [CI]: 52.4-81.4). Blood samples and ticks were tested using polymerase chain reaction to detect spotted fever group (SFG) rickettsiae. One blood sample was positive for Rickettsia parkeri (2.3%; 95% CI: 0.06-12.3). Ticks were collected from 10 dogs and were identified as Amblyomma tigrinum (n = 44) and Rhipicephalus sanguineus (n = 1). All A. tigrinum ticks were collected from resident dogs from the rural areas of Cochabamba, whereas R. sanguineus was from a dog originating from Santa Cruz. Of 42 DNA samples extracted from ticks, 23 (54.8%; 95% CI: 38.7-70.1) were polymerase chain reaction positive for Rickettsia spp. Sequencing analysis identified 22 samples as R. parkeri and one as Rickettsia aeschlimannii. Positive ticks (all A. tigrinum) were collected from six dogs, all of which were seropositive. This is the first report of SFG rickettsiae in A. tigrinum, suggesting that this tick-like others species in the Amblyomma maculatum group--may play a role in the biological cycle of Ri. parkeri. The high infection prevalence of SFG rickettsiae in ticks and the even higher seroprevalence in dogs suggest an active circulation of agents of rickettsiosis in the study area, although there are no confirmed cases of infection in humans. Our study supports the use of canine serology as risk indicator for SF rickettsioses.